The objective of this research is to determine the mechanisms for protein export in Bacillus subtilis. This project seeks to identify and characterize intermediate forms of extracellular alpha-amylase, alkaline and neutral protease and factors in the export process involved in processing of intermediate enzyme forms. Experiments are planned to search for, isolate and characterize pre-enzymes containing NH2-terminal peptide extensions (signal peptides) and membrane-bound forms of alpha-amylase, alkaline and neutral protease. Small amounts of intermediate exoenzyme forms will be obtained by immunoprecipitation from in vivo or in vitro syntheses using radioactive amino acids to obtain radioactive proteins suitable for sequencing. Mutant methodology and experiments with protoplasts will be used to detect processing enzymes. Processing enzymes will be isolated, purified and characterized.